PROJECT SUMMARY Illegal use of psychostimulants, such as cocaine and methamphetamine, incurs huge socioeconomic costs worldwide. Despite advances in our understanding of the neural mechanisms that mediate substance abuse and addiction, little is known about genetic risk factors. Genetic studies in human populations are confounded by genetic and environmental heterogeneity and uncontrolled variation in drug exposure. Drosophila melanogaster presents a powerful model system to study the genetic underpinnings of drug susceptibility, since the genetic background and environment, including exposure to drugs, can be controlled precisely. Many effects of psychostimulants on people are replicated in flies and are likely mediated through the D. melanogaster dopamine transporter, which has recently been crystallized and shown to bind cocaine and metamphetamine. Here, we propose to capitalize on natural genetic and phenotypic variation in an outbred advanced intercross population derived from a population of 205 inbred wild-derived lines of D. melanogaster with sequenced and well annotated genomes, the D. melanogaster Genetic Reference Panel (DGRP), to explore the genetic architectures that underlie variation in voluntary consumption of cocaine and methamphetamine. We will perform extreme QTL mapping and artificial selection combined with DNA and RNA sequencing as two complementary genetic mapping strategies to identify naturally occurring variants affecting consumption of cocaine and methamphetamine. We will use segregating variation in the DGRP as well as mutations and RNAi constructs in co-isogenic backgrounds to perform functional validation experiments of the candidate genes and variants identified in each approach. We will focus on increased consumption of cocaine and methamphetamine, since consumption is genetically variable in the DGRP, we have previously derived selection lines for food consumption under control conditions, and preliminary data show that some DGRP lines voluntarily consume larger amounts of cocaine or metamphetamine than a 4% sucrose control solution, suggesting it may be possible to develop a Drosophila model for complex behaviors related to addiction. The specific aims of this application are: (1) To identify genetic variants in a DGRP-derived advanced intercross population associated with increased consumption of cocaine and methamphetamine using extreme QTL mapping by DNA sequencing of pools of extreme individuals and randomly selected controls. (2) To create lines enriched for variants associated with increased consumption of cocaine and methamphetamine by long-term artificial selection. (3) To functionally validate variants and genes associated with increased consumption of cocaine and methamphetamine from Specific Aims 1 and 2. orthologs in humans. R We esults from the proposed experiments under the U01 mechanism will will focus on testing evolutionarily conserved candidate genes with culminate in a wealth of novel information that can guide future genetic studies in mammals, including humans.